Current contrasting and labelling methods for optical imaging are extensively used in light absorption, optical reflection and molecular fluorescence. Such optical imaging techniques are often used for applications such as medical diagnosis, civil security, mining exploration, etc. Fluorescence labelling is used in many different applications such as, for example, automated DNA sequencing.
Recently, there has been a growing interest in the development of optical imaging techniques in which the high contrast is molecular specific and based on molecular vibrations. The challenge is to obtain an unambiguous molecular detection without loss of sensitivity. Raman spectroscopy is among the most powerful techniques available for the identification and analysis of molecular vibrations, but it lacks sensitivity relative to other spectroscopic techniques. As a result, Raman imaging and the use of Raman molecular probes are rarely found in commercial applications.
Raman Sensitivity
The sensitivity of various imaging techniques can be compared by considering the cross section required to observe light scattering. In Raman spectroscopy, the intensity, I, (in photons/s/cm2) of scattered light for a molecule is proportional to the scattering cross section per molecule σR and the intensity of the incident light Io, according to the relation I=σRIo. For Raman spectroscopy, σR is between 10−29 and 10−32 cm2, while the equivalent fluorescence and optical absorption cross sections are on the order of 10−19 to 10−18 and 10−29 to 10−32 cm2, respectively (S. Nie and S. R. Emory, “Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering”, Science 275 1 102-1106 (1997)). There are therefore approximately more than twelve orders of magnitude difference between the relative efficiencies of the Raman process and those of optical absorption or fluorescence. Raman spectroscopy benefits, however, from high laser intensities, which compensate for the low efficiency of the scattering process to make this analytical technique more accessible. Nevertheless, the low sensitivity remains a problem for Raman imaging. In addition, the use of high intensity excitation lasers can alter the samples being examined due to localized heating. In such cases, the acquisition of a Raman image is done by sweeping the light-emitting probe point by point with a reduced intensity to avoid the heating, which makes the acquisition time-consuming and inefficient. Being much more sensitive, fluorescence and absorption/reflection have been heretofore the techniques of choice for optical imaging [V. Ntziachristos, Fluorescence molecular Imaging, Annual Review of Biomedical Engineering, Vol. 8: 1-33 (2006)].
Use of Optical Probes for Chemical Analysis
Chemical analysis is possible in absorption and fluorescence spectroscopy, but absorption or fluorescent emission bands are wide and imprecise. However, the optical contrasts in absorption are generally weak for materials with similar transparencies, and most molecules are not or only weakly fluorescent. Thus, it is often necessary to add optical dyes to the samples. There is a wide range of optical dyes or fluorophores available on the market, and these are frequently used as contrast agents or molecular probes. This practice is also commonly used to improve the contrasts in photoacoustic imaging [A. De La Zerda et al. “Carbon nanotubes as photoacoustic molecular imaging agents in living mice” Nat. Nanotechnol. Vol. 3, No. 9, 557-562 (2008)]. Since these contrast agents have very wide absorption or emission bands, it is, however, difficult to mix multiple contrast agents such as these and preserve a clean wavelength signature for each.
On the other hand, a highly specific molecular contrast is possible with Raman and infrared spectroscopy because they provide information on the vibrational transitions of the molecules (from 100 cm−1 to 6000 cm−1) and present a series of very narrow spectral bands (generally less than 5 cm−1). Each molecule or solid possesses a rich spectrum of vibrational transitions, and their Raman and infrared spectra give precisely this information; the vibrational spectrum being somewhat like a “fingerprint” of the molecule.
Raman and infrared absorption are thus very powerful techniques for chemical analysis, but the weakness of each is the strength of the other. Infrared absorption offers a good sensitivity (σR˜10−21 cm2) relative to Raman (σR˜10−29 cm2), but this efficiency is mitigated by the poor sensitivities of optical detectors in the infrared region. Raman operates instead in the visible range (400-800 nm) where detectors of the type Si CCD are very efficient and sensitive (only a few photons are needed for signal detection). Moreover, the spatial resolution is poor in infrared and excellent in Raman because the resolution limit depends on wavelength (the limit of resolution is ˜λ/2 according to the Rayleigh criterion), which is relatively long for infrared (λ˜30 μm) and short for Raman (λ=400-600 nm). Applications using Raman would be ideal, but the problem arises with the cross sections of Raman scattering, which are too weak to be useful in optical imaging or molecular marking.
Amplified Raman Probes
Solutions have been proposed to attempt to improve the sensitivity of molecular detection in Raman scattering.
For example, it has been observed that there can be an amplification of a Raman signal when probe molecules are in proximity to metal particles or rough surfaces [S. Nie and S. R. Emory, “Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering”, Science, 275, 1102-1106 (1997)]. This signal enhancement results from a local amplification of the electric field in the immediate vicinity of metallic objects that permits a significant improvement in the Raman scattering cross section. These gains on the Raman signal are generally referred to as “Surface-Enhanced Raman Spectroscopy” (SERS) or “Surface-Enhanced Resonance Raman Spectroscopy” (SERRS). There are a large number of SERS or SERRS probes prepared using metallic particles or metallic surfaces linked chemically or physically with one or more dye molecules. These probes linked to resonant molecules and the possible signal enhancement with these probes can reach ˜1014. These probes are, however, difficult to prepare, are often toxic (for in-vivo applications) and require preparations or syntheses that are expensive and difficult to reproduce. Moreover, it is difficult to extend these effects to probes or tags having nanometric dimensions.
It has also been observed that the Raman scattering cross section of carbon nanotubes is exceptional, on the order of σR˜10−21 cm2[A. Jorio et al. “Structural (n, m) Determination of Isolated Single-Wall Carbon Nanotubes by Resonant Raman Scattering”, Phys. Rev. Lett., Vol. 86, No. 6, 1118-1121 (2001); J. E. Bohn et al. 25 “Estimating the Raman cross sections of single carbon nanotubes”, ACS Nano 4 (6), 3466-3470 (2010)]. This property is quasi-unique in the world of nanostructures and is comparable to the resonant Raman cross sections of an aggregate of molecules assembled by stacking in a large structure. The physics of the Raman scattering phenomenon in nanotubes is fairly well understood, because it relates to a resonant process and the object is made up of a number of well-organized atoms (i.e., the nanotube is large relative to a molecule). The nanotube is therefore, in itself, a very interesting Raman probe but, in practice, nanotubes tend to be provided as a mix of different nanotubes, and it is difficult to obtain a sample of nanotubes of the same type. To be useful as a probe, it is necessary to sort the nanotubes by chirality or different isotope composition. [Z. Liu, S. Tabakman, S. Sherlock, X. Li, Z. Chen, K. Jiang, S. Fan, and H. Dai, Multiplexed Five-Color Molecular Imaging of Cancer Cells and Tumor Tissues with Carbon Nanotube Raman Tags in the Near-Infrared Nano Res 3: 222-233(2010)] Only two isotopes of carbon (C12 and C13) are available, which imposes an important limitation to the diversity of the library. Although methods exist for separating nanotubes, they are expensive and produce a very small amount of material. Moreover, a chemical functionalization of a nanotube generally diminishes its Raman signal.
The development of a Raman probe based on carbon nanotubes is interesting, but it remains difficult to use its Raman signal for a clear identification of the probe.
Highly Raman-Active Molecules
Even if the Raman scattering is inefficient, there exists a large number of molecules that are Raman-active. To obtain a strong signal, a high concentration of molecules is necessary in the analysis zone. Despite this limitation, Raman spectroscopy allows the characterization of molecules present in a particular environment. A large set of molecular dyes are strongly active in Raman and this is possible because of their resonance in the visible spectrum. Well known examples include conjugated molecules such as β-carotene, pyridine and rhodamine 6-G. The scattering in these molecules involves resonant Raman and the cross section (by molecule) can attain up to 10−24 to 10−25 cm2, at the wavelength of the resonance. [S. Shim, C. M. Stuart, and R. A. Mathies, Resonance Raman Cross-Sections and Vibronic Analysis of Rhodamine 6G from Broadband Stimulated Raman Spectroscopy, ChemPhysChem 9, 697-69 (2008)] Despite this, the cross sections are still weak with regard to that which would be necessary for molecular marking or tagging applications. In such a case, it would not be possible to detect a single molecule with resonant Raman, and multiple molecules are necessary in the analysis zone to obtain an acceptable signal. In addition, most of these molecules are unstable under the influence of a high laser intensity and present a luminescence that can diminish or mask a Raman signal.
There is therefore a need for new probes appropriate for optical imaging or molecular marking that have a high sensitivity and can be obtained by relative simple and low-cost preparation methods.
There is a need for new probes appropriate for optical imaging or molecular marking that use Raman scattering, that have a diminished fluorescence and that allow a strong and distinct Raman signal.
There is a need for new probes appropriate for optical imaging and molecular marking and for which a high concentration of probes is not necessary to obtain an acceptable signal.
There is a need for new probes appropriate for optical imaging or molecular marking and based on Raman scattering that are individually detectable and identifiable.
There is a need for new probes appropriate for optical imaging and molecular marking and based on Raman scattering that allow multiple, different dyes to be used simultaneously while each maintaining its specific wavelength signature.